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Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this “restriction” of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3.The present protocol (Nair et al., 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner.
The protocol described here is mainly adapted from that described by Iwatani et al. (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.

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In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein
APOBEC3 蛋白质胞苷脱氨酶活性的体外检测

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Smita Nair
Smita NairAffiliation: Center for Cancer Research, National Cancer Institute, Frederick, USA
Present address: London School of Hygiene and Tropical Medicine, Faculty of Infectious and Tropical Diseases, London, UK
Bio-protocol author page: a1730
 and Alan Rein
Alan ReinAffiliation: Center for Cancer Research, National Cancer Institute, Frederick, USA
For correspondence: reina@mail.nih.gov
Bio-protocol author page: a1731
Vol 4, Iss 20, 10/20/2014, 3954 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1266

[Abstract] Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this “restriction” of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3.The present protocol (Nair et al., 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner.
The protocol described here is mainly adapted from that described by Iwatani et al. (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.
Keywords: APOBEC3(APOBEC3蛋白), Retrovirus restriction(逆转录病毒限制), Cytidine deaminase(胞苷脱氨酶), In vitro deaminase assay(体外酶法)

[Abstract]

Materials and Reagents

  1. 5’ N-AlexaFluor488-labeled oligodeoxynucleotide (custom synthesized from Integrated DNA Technologies)
    Example: 5' -/5Alex488N/ATA ATA ATA ATA ATA ATA ATA ATA TCC ATA ATA ATA ATA ATA ATA ATA-3’, PAGE purified, 250 nmol synthesis scale (see Recipes) (while a 48-base oligodeoxynucleotide is described here, both longer and shorter oligodeoxynucleotides could undoubtedly be used).
  2. Nuclease-free or autoclaved sterile water
  3. 1 U/µl Uracil DNA glycosylase (Thermo Fisher Scientific, catalog number: EN0361 )
  4. 0.6 N NaOH
  5. 2x RNA loading dye (Thermo Fisher Scientific, catalog number: R0641 )
  6. 10 bp DNA ladder (Life Technologies, catalog number: 10821-015 )
  7. Tris (Sigma-Aldrich)
  8. NaCl (Sigma-Aldrich)
  9. DTT (Sigma-Aldrich)
  10. Novex® TBE-UREA gel (Life Technologies, catalog number: EC6885BOX/EC68852BOX )
  11. Boric acid
  12. EDTA
  13. 10x deaminase buffer (see Recipes)
  14. 10x Uracil DNA glycosylase reaction buffer (see Recipes)
  15. 10x Tris borate EDTA (TBE) buffer (see Recipes)
  16. N-AlexaFluor labelled oligodeoxynucleotides (see Recipes)

Equipment

  1. RNase- and DNase-free 0.5 ml microcentrifuge tubes
  2. 37 °C water bath
  3. Heating Block set to 95 °C
  4. XCellSureLock® Mini-Cell Electrophoresis apparatus (Life Technologies, catalog number: EI0001 )
  5. Powerpac Basic Power Supply (Bio-Rad Laboratories, catalog number: 164-5050 )
  6. Typhoon Imager (Trio+, variable mode imager) (GE Healthcare, catalog number: 2340730 )

Procedure

  1. Set up 10 µl deaminase assay reactions in 0.5 ml microcentrifuge tubes as follows. First prepare a deaminase assay Master Mix, with enough volume for all of the reactions plus some excess. The table below gives the recipe for a Master Mix for 10 reactions, although enough Master Mix (99 µl) is prepared for 11 reactions:

    Deaminase assay components
    Amount per reaction
    Master mix
    Water
    7.5 µl
    82.5 µl
    10x deaminase buffer
    1 µl
    11 µl
    Fluorophore-labeled oligonucleotide substrate (10 pmol/µl)
    0.5 µl
    5.5 µl

    Dispense 9 µl of the Master Mix into each tube; then add 1 µl of the protein preparation to be assayed to each tube. In our experience, approximately 0.25 µg of mouse APOBEC3 is sufficient for deamination of a large fraction of the oligodeoxynucleotide substrate in this assay. (A no-protein, “blank” control reaction should also be prepared to which protein buffer, instead of protein, is added.)
    Gently mix the reactants by pipetting up and down, and if required, give a quick spin to collect all the contents at the bottom of the tube. Incubate the reaction in a 37 °C water bath for 2 h.
    Note: During the 2 h incubation at 37 °C, centrifuge the contents of the tubes at low speed occasionally as moisture tends to accumulate at the tube caps owing to evaporation.
  2. Prepare UDG Master Mix as indicated in the table below. For example, if 10 assays are being performed, prepare 55 µl UDG Master Mix, i.e., enough for 5 µl/reaction + 5 µl excess volume:

    UDG enzyme mix
    Amount per reaction
    Master mix
    Water
    2.75 µl
    30.25 µl
    10x UDG buffer
    1.5 µl
    16.5 µl
    UDG enzyme (1 U/µl)
    0.75 µl
    8.25 µl

    Add 5 µl of this UDG Master Mix to the 10 µl deaminase assay reaction tubes from step 1. Gently mix the reactants and incubate in the 37 °C water bath for 45 min.
  3. Following the 45 min incubation, add 5 µl of 0.6 N NaOH to each tube and incubate further at 37 °C for 20 min.
  4. Add an equal volume (20 µl) of 2x RNA loading dye to each tube, heat at 95 °C for 2-3 min, and place on ice immediately.
  5. Load 15 µl of each sample onto a 15% TBE-UREA PAGE gel and resolve the cleaved and uncleaved products by electrophoresis at 150 volts for 3 h at room temperature (25 °C) in 1x TBE buffer. (Before loading the samples, flush the wells with running buffer using a syringe fitted with a needle. This removes residual urea or acrylamide from the wells and minimizes the probability of samples smearing on the gel.)
  6. In order to visualize the uncleaved substrate and cleaved product(s), remove the gel carefully from the cast, slice away the sealing gel end at the bottom, and place the gel onto a clean glass plate. Carefully place a cling film/saran wrap on the gel; remove any air bubbles between the gel and the film by gently rolling a roller (supplied with the gel apparatus) or a Pasteur pipette over it. Air bubbles tend to interfere with the fluorescence imaging. A trick to avoid air bubbles and to keep the gel from drying and breaking is to generously put several drops of water on the gel before placing the cling film.
  7. Place the gel on the fluorescence reader, gel side facing the scanner and glass side up. The scanner should be set for AlexaFluor 488 (excitation 495 nm, Emission 519 nm), PMT 600, sensitivity normal, and scanning speed 200 microns.

Representative data

  1. A representative gel is shown in Figure 1. In general we have found this assay to be extremely robust and reproducible.


    Figure 1. Mouse APOBEC3 was tested with two 48-base oligodeoxynucleotides.
    The first (left panel) was composed entirely of A and T residues, except for residues 25-27, which were TTC, and which was labeled with AlexaFluor 488 at its 5’ end. The oligonucleotide was incubated with no protein (first lane) and with mouse APOBEC3 produced in Sf9 cells (second and third lanes). The second lane shows the results of this incubation, followed by the full assay protocol described here, while the third lane shows the results when UDG was omitted from the assay. The right panel shows a similar assay, but the oligonucleotide contained, in addition to A and T residues, TCCC at residues 21-24 and TTCC at residues 28-31.

Notes

  1. While the assay we have described here monitors the deamination of a substrate oligodeoxynucleotide with a fluorophore at its 5’ end, many investigators have used 5’- 32P-labeled oligodeoxynucleotides instead, measuring the cleavage by autoradiography rather than fluorescence imaging. Some researchers may find this more convenient or economical than the use of fluorescence. However, the principles and most of the procedures used in the two methods are identical.
  2. As a positive control on the assay, one can design an oligonucleotide identical to the substrate (Materials and Reagents) except that it contains uracil rather than cytosine (5' - /5Alex488N/ ATA ATA ATA ATA ATA ATA ATA ATA TUU ATA ATA ATA ATA ATA ATA ATA 3’). This should be completely cleaved in the UDG-alkali treatments; if it is not, this is an indication that the treatments are not successful in inducing cleavage at all deoxyuridines in the oligodeoxynucleotides.
  3. The protocol here (“Procedure”, text of step 1) calls for the addition of 1 µl of the protein preparation to be assayed for deaminase activity. Our preparations (Nair et al., 2014) were partially purified from Sf9 insect cells and typically contained 0.5-1 µg of protein per µl.

Recipes

  1. 10x deaminase buffer
    100 mM Tris.HCl (pH 8.0)
    500 mM NaCl
    10 mM DTT
    Note: Tris, NaCl and DTT were all procured from Sigma Aldrich. Stock solutions of 1 M Tris HCl (pH 8.0) and 5 M NaCl were autoclaved and stored at RT, while 1 M DTT solution was freshly made and was stored at -20 °C.

    10x deaminase buffer (1 ml)
    Volume (µl)
    1 M Tris.HCl (pH 8.0)
    100
    5 M NaCl
    100
    1 M DTT
    10
    Water
    790

    This buffer should be stored at -20 °C.
  2. 10x Uracil DNA glycosylase reaction buffer
    200 mM Tris-HCl (pH 8.2 at 25 °C)
    10 mM EDTA
    100 mM NaCl
    Supplied with Uracil DNA glycosylase
  3. 10x Tris borate EDTA (TBE) buffer
    108 g Tris base
    55 g boric acid
    40 ml 0.5 M EDTA (pH ~8.3)
  4. N-AlexaFluor labelled oligodeoxynucleotides
    These are supplied as dried powder/pellet/precipitate. Dissolve in nuclease-free or autoclaved sterile water to a final concentration of 1 nmol/µl stock. Stored at -20 °C, in dark tubes. In order to avoid repeated freezing and thawing of the substrate it is advisable to make several aliquots of 50 µl each of a working concentration of 10 pmol/µl and store at -20 °C in dark tubes. Although the yield of the oligodeoxynucleotides is generally only 2-5% of the scale of synthesis, it may well be sufficient for the assays as each assay reaction only consumes 5 pmol or even less.

Acknowledgements

We thank Jennifer Miller and Jason Rausch for help with the assay. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

References

  1. Iwatani, Y., Takeuchi, H., Strebel, K. and Levin, J. G. (2006). Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J Virol 80(12): 5992-6002.
  2. Nair, S., Sanchez-Martinez, S., Ji, X. and Rein, A. (2014). Biochemical and biological studies of mouse APOBEC3. J Virol 88(7): 3850-3860.

材料和试剂

  1. 5'N-AlexaFluor488-标记的寡脱氧核苷酸(由Integrated DNA Technologies定制合成)
    实施例:5'-/5Alex488N/ATA ATA ATA ATA ATA ATA ATA TCC ATA ATA ATA ATA ATA ATA-3',PAGE纯化,250nmol合成规模(参见Recipes)(尽管这里描述了48-碱基寡脱氧核苷酸, 毫无疑问,可以使用更长和更短的寡脱氧核苷酸)
  2. 无核酸酶或高压灭菌的无菌水
  3. 1U /μl尿嘧啶DNA糖基化酶(Thermo Fisher Scientific,目录号:EN0361)
  4. 0.6 N NaOH
  5. 2x RNA加载染料(Thermo Fisher Scientific,目录号:R0641)
  6. 10bp DNA梯(Life Technologies,目录号:10821-015)
  7. Tris(Sigma-Aldrich)
  8. NaCl(Sigma-Aldrich)
  9. DTT(Sigma-Aldrich)
  10. Novex TBE-UREA凝胶(Life Technologies,目录号:EC6885BOX/EC68852BOX)
  11. 硼酸
  12. EDTA
  13. 10x脱氨酶缓冲液(参见配方)
  14. 10x尿嘧啶DNA糖基化酶反应缓冲液(参见配方)
  15. 10x Tris硼酸盐EDTA(TBE)缓冲液(见配方)
  16. N-AlexaFluor标记的寡脱氧核苷酸(参见Recipes)

设备

  1. 无RNase和DNase的0.5ml微量离心管
  2. 37°C水浴
  3. 加热块设置为95°C
  4. XCell SureLock Mini-Cell Electrophoresis apparatus(Life Technologies,目录号:EI0001)
  5. Powerpac基本电源(Bio-Rad Laboratories,目录号:164-5050)
  6. Typhoon Imager(Trio +,可变模式成像仪)(GE Healthcare,目录号:2340730)

程序

  1. 如下在0.5ml微量离心管中设置10μl脱氨酶测定反应。 首先准备脱氨酶测定主混合物,具有足够的体积用于所有反应加上一些过量。 下表给出了10个反应的主混合物的配方,尽管为11个反应制备了足够的主混合物(99μl):

    脱氨酶测定组件
    每次反应金额
    主混音

    7.5μl
    82.5微升
    10x脱氨酶缓冲液
    1微升
    11微升
    荧光标记的寡核苷酸底物(10pmol /μl)
    0.5μl
    5.5微升

    将9μl主混合物分配到每个管中;然后向每个管中加入1μl待测定的蛋白质制备物。根据我们的经验,在该测定中,大约0.25μg的小鼠APOBEC3足以使大部分寡脱氧核苷酸底物脱氨基。 (无蛋白,"空白"对照反应也应制备添加蛋白缓冲液,而不是蛋白质。)
    通过上下吹吸轻轻混合反应物,如果需要,快速旋转以收集管底部的所有内容物。将反应在37℃水浴中孵育2小时 注意:在37°C孵育2小时时,偶尔会以低速离心管中的内容物,因为水分会由于蒸发而积聚在管帽上。
  2. 按下表所示制备UDG主混合物。例如,如果进行10次测定,则准备55μlUDG主混合物,即足以用于5μl/反应+5μl过量体积:

    UDG酶组合
    每次反应金额
    主混音

    2.75微升
    30.25微升
    10x UDG缓冲区
    1.5μl
    16.5微升
    UDG酶(1U /μl)
    0.75μl
    8.25μl

    将5μl该UDG主混合物加入到步骤1的10μl脱氨酶测定反应管中。轻轻混合反应物,并在37℃水浴中孵育45分钟。
  3. 在45分钟孵育后,向每个管中加入5μl的0.6N NaOH,并在37℃下进一步温育20分钟。
  4. 向每个管中加入等体积(20μl)2x RNA加载染料,在95℃加热2-3分钟,立即置于冰上。
  5. 将15μl每种样品加载到15%TBE-UREA PAGE凝胶上,并通过在150伏在室温(25℃)下在1x TBE缓冲液中电泳3小时来分解切割和未切割的产物。 (在装载样品前,使用带针头的注射器用运行缓冲液冲洗孔,从孔中除去残留的尿素或丙烯酰胺,并使样品在凝胶上涂抹的概率最小)。
  6. 为了可视化未裂解的底物和裂解产物,从铸件中小心地除去凝胶,在底部切下密封凝胶末端,并将凝胶置于干净的玻璃板上。小心地在胶上放置保鲜膜/通过轻轻地滚动辊(与凝胶装置一起提供)或巴斯德吸管在其上除去凝胶和膜之间的任何气泡。气泡倾向于干扰荧光成像。避免气泡和防止凝胶干燥和破裂的一个窍门是在放置保鲜膜之前,将几滴水大量地放在凝胶上。
  7. 将凝胶放在荧光读数器上,凝胶侧面向扫描仪,玻璃面朝上。扫描仪应设置为AlexaFluor 488(激发495 nm,发射519 nm),PMT 600,灵敏度正常,扫描速度200微米。

代表数据

  1. 代表性的凝胶如图1所示。一般来说,我们发现这种测定是非常稳定和可重复的。


    图1.用两个48-碱基寡脱氧核苷酸测试小鼠APOBEC3。第一个(左图)完全由A和T残基组成,除了残基25-27,其是TTC,并且用AlexaFluor标记488在其5'端。将寡核苷酸与无蛋白(第一泳道)和在Sf9细胞中产生的小鼠APOBEC3(第二和第三泳道)一起温育。第二泳道显示了该温育的结果,随后是本文所述的完全测定方案,而第三泳道显示了当从测定中省略UDG时的结果。右图显示了相似的测定,但是除了A和T残基之外,寡核苷酸还包含残基21-24的TCCC和残基28-31的TTCC。

笔记

  1. 尽管我们在此描述的测定法监测了在其5'末端具有荧光团的底物寡脱氧核苷酸的脱氨基,但是许多研究者已经使用5'→32 P标记的寡脱氧核苷酸,而通过放射自显影测量切割,比荧光成像。一些研究人员可能发现这比使用荧光更方便或更经济。但是,两种方法中使用的原理和大多数过程是相同的。
  2. 作为测定的阳性对照,可以设计与底物(材料和试剂)相同的寡核苷酸,除了其含有尿嘧啶而不是胞嘧啶(5' -/5Alex488N/ATA ATA ATA ATA ATA ATA ATA ATA TUU ATA ATA ATA ATA ATA ATA ATA 3')。这应该在UDG碱处理中完全裂解;如果不是,这表明治疗不能成功诱导寡脱氧核苷酸中所有脱氧尿苷的切割。
  3. 这里的方案("Procedure",步骤1的文本)要求加入1μl待测定脱氨酶活性的蛋白质制剂。我们的制备(Nair等人,2014)从Sf9昆虫细胞中部分纯化,通常每μl含有0.5-1μg蛋白质。

食谱

  1. 10x脱氨酶缓冲液
    100mM Tris.HCl(pH 8.0)
    500 mM NaCl
    10 mM DTT
    注意:Tris,NaCl和DTT均购自Sigma Aldrich。 将1M Tris HCl(pH8.0)和5M NaCl的储备溶液高压灭菌并在室温下储存,同时新鲜制备1M DTT溶液并储存在-20℃。

    10x脱氨酶缓冲液(1ml) 体积(μl)
    1M Tris缓冲液(pH8.0) 100
    5 M NaCl
    100
    1 M DTT
    10

    790

    此缓冲液应储存在-20°C
  2. 10x尿嘧啶DNA糖基化酶反应缓冲液
    200mM Tris-HCl(pH8.2,25℃) 10 mM EDTA
    100 mM NaCl
    供应尿嘧啶DNA糖基化酶
  3. 10x Tris硼酸盐EDTA(TBE)缓冲液 108克Tris碱
    55克硼酸 40ml 0.5M EDTA(pH〜8.3)
  4. N-AlexaFluor标记的寡脱氧核苷酸 这些以干燥的粉末/丸/沉淀物形式提供。 溶于无核酸酶或高压灭菌的无菌水至终浓度为1nmol /μl原液。 储存于-20°C,在黑暗的试管中。 为了避免底物的重复冷冻和解冻,建议制备几个等分试样,每个50μl的工作浓度为10pmol/μl,并在-20℃下在暗管中储存。 尽管寡脱氧核苷酸的产量通常仅为量级的2-5% 合成,对于测定可能是足够的,因为每个测定反应仅消耗5pmol或甚至更少

致谢

我们感谢Jennifer Miller和Jason Rausch对测定的帮助。这项工作是由国家卫生研究院,国家癌症研究所,癌症研究中心的校内研究计划的支持。

参考文献

  1. Iwatani,Y.,Takeuchi,H.,Strebel,K。和Levin,J.G。(2006)。 高纯度,催化活性人类APOBEC3G的生物化学活性:与抗病毒效应的相关性。 em> J Virol 80(12):5992-6002。
  2. Nair,S.,Sanchez-Martinez,S.,Ji,X。和Rein,A。(2014)。 小鼠APOBEC3的生物化学和生物学研究 J Virol 88(7):3850-3860。
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How to cite this protocol: Nair, S. and Rein, A. (2014). In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein. Bio-protocol 4(20): e1266. DOI: 10.21769/BioProtoc.1266; Full Text



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(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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